Structural and functional analysis of virus siRNAs

siRNA The RNA silencing based antiviral plant response is one of the best studied antiviral strategies in plants. The key element of RNA silencing based antiviral strategies is the virus derived small interfering RNA (vsiRNA), which guides the RNA induced silencing complex (RISC) to target viral genomes in plants and invertebrates. siRNAs are processed from double-stranded RNAs (dsRNA) or structured single-stranded RNAs (ssRNAs) by RNase III-like enzymes such as DICER (in plants there are several Dicer-like genes). siRNAs guide the sequence-specific inactivation of target mRNAs by RISC. Plant RNA viruses are strong inducers as well as targets of RNA silencing and high levels of vsiRNAs accumulate during the viral infection. However, despite of the extensive studies of siRNA biogenesis the origin of plant viral siRNA is still not understood. vsiRNAs are thought to be processed from ds viral RNA replication intermediates, local self-complementary ds regions of the viral genome or through the action of RNA-dependent RNA polymerases on viral RNA templates. In plants two distinct classes of vsiRNAs have been identified: the primary siRNAs, which result from DCL mediated cleavage of an initial trigger RNA, and secondary siRNAs, whose biogenesis requires an RDR enzyme.

Researchers profiled Cymbidium ringspot virus (CymRSV) derived short RNAs using three different methods. Profiling of viral short interfering RNAs revealed a different sequence bias for the 454 and Solexa high-throughput sequencing platforms. They found that viral short RNAs are primarily produced from the positive strand of the virus and produced with very different frequency along the viral genome. The hybridisation approach showed that the profile of viral short RNAs is determined by the virus itself because the profiles were the same in different species and it also showed that the process was RDR6 independent. These results suggest that CymRSV short RNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase. Regions from the viral genome that are not complementary to highly abundant viral short RNAs were targeted in the plant just as efficiently as regions recognised by abundant short RNAs.

Structural and Functional Analysis of Viral siRNAs. 2010 PLoS Pathog 6(4): e1000838. doi:10.1371/journal.ppat.1000838
A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.

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